Malaria is an infectious disease caused by Plasmodium parasites. There are 5 species which infect humans:                1. Plasmodium falciparum (the most virulent)        2. Plasmodium vivax         3. Plasmodium malariae         4. Plasmodium ovale         5. Plasmodium knowlesi (primarily infect monkeys but also humans)        
P. falciparum, P. malariae and P. knowlesi can invade and grow in young and old red blood cells, but P. vivax and P. ovale can only invade young red cells (reticulocytes). P. falciparum and P. vivax are the most abundant forms in Thailand. Female Anopheles dirus and Anopheles minimus are the major mosquito species that transmit malaria in Thailand.
Throughout malaria endemic areas of the World, malaria parasites have developed resistance to most available anti-malarial drugs. There is thus an urgent need for new anti-malarial to counter resistance. Development of anti-malarial drugs is based on empirical screening of natural products and rational drug design against known drug targets. Plasmodium dihydrofolate reductase-thymidylate synthase (DHFR-TS) is one of the best characterized targets and has gained a lot of interest as a target for rational drug design. DHFR-TS is a bifunctional enzyme in which the DHFR and TS enzymatic moieties are connected by a junction region (JR). Mutations in the DHFR domain have been found to associate with antifolate resistance. Rational drug design against DHFR is greatly assisted by the availability of several high-resolution crystal structures of this enzyme (including antifolate-resistant variants) in complex with inhibitors such as pyrimethamine, cycloguanil and WR99210. Indeed, this enzyme is also an important drug target for other infectious diseases. In bacteria, the DHFR and TS enzymes are encoded by the folA and thyA genes, respectively. Trimethoprim is an effective inhibitor against the bacterial folA product.
In order to evaluate the anti-malarial activity of compounds, in vitro anti-malarial screening using malaria parasites grown in human red blood cells has been widely employed. The parasites are cultivated in red blood cells with culture media containing human serum. Routine changing of culture media and supplying of new blood cells for the parasites is needed. Moreover, evaluation of drug efficacy requires microscopic, fluorescent, or radioactive methods for enumerating/measuring parasite growth. These requirements are a hindrance to high throughput screening and limit anti-malarial screening to centers with malaria culture systems in place. For target-based anti-malarial screening (which includes antifolates), surrogate models are useful alternatives when malaria culture facilities are not available. Bacterial surrogate models have been employed for antifolate anti-malarial screening in which a folA deficient bacterial cell is complemented by Plasmodium DHFR. The bacterial surrogate is made folA deficient either by adding trimethoprim (chemical knockout through inhibition of the host folA enzyme product) or using the PA414 strain, a folA genetic knockout strain. However, anti-malarial drug efficacy evaluated by these methods correlates poorly with conventional anti-malarial screening methods that employ cultured parasites. This may largely be due to off-target interference by trimethoprim and the poor growth rate of PA414, respectively.
This invention entails an Escherichia coli strain whose thyA and folA genes were disrupted using genetic knockout. We evaluated the use of this invention as a host for screening DHFR inhibitors against Plasmodium malaria and other parasites. This tool, thyA folA KO E. coli, is easy and convenient to use. It gives quick and reliable results which correlate well with the conventional anti-malarial screening system. With this tool, it is feasible to perform antifolate assay against malaria and other parasitic diseases in a laboratory with facilities for bacterial cell culture, which more are widely available than parasite culture facilities.